ANTHER CULTURE

International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705
www.rsisinternational.org Page 1
Production of Haploids Plants from Anther Culture of
Musa Paradisiaca cv. ‘Puttabale’
K. Girish Kumar1
, V. Krishna2
*, Venkatesh3
, R. Shashikumar4
, H. S. Arunodaya5
1, 2, 4, 5
Department of P. G. Studies and Research in Biotechnology, Bioscience Block, Kuvempu University, Jnana Sahyadri,
Shankaraghatta‐577 451, Shivamogga (Dist.), Karnataka, India.
3
Department of Biochemistry Indian Institute of Science Bangalore. Karnataka, India
* Corresponding author: V. Krishna
Abstract: Haploid plants were regenerated from the anther
callus of banana Musa paradisiaca (AB) cv. Puttabale. The
highest frequency of callus induction (90%) was observed
at the concentration of 3mg/l 2, 4-D . After 20 days of
incubation organization of embyroids were organised
from the callus mass. Interaction of 4mg/l BAP and 0.4
mg/l IAA provoked shoot growth of the embryoids and
well organised roots were developed at the concentration
of 0.6 mg/l NAA and the media was agumented with 0.2%
activated charcoal. Flow cytometry study was carried out
to analyse the DNA content of the regenerated haploid
plants. The results of the investigation reported the
efficient production of haploid plants from the anther
culture.
Key words: Musa paradisica, puttabale, anther culture,
haploid plants
I. INTRODUCTION
anana and plantain (Musa spp.) are the world’s major
food crop for millions of people. Many breeders
improved the quality by manipulating the ploidy of this crop
(Pillay et al., 2003). But the conventional breeding method is
laborious and took more generations for the development of
haploid plants. The production of haploids through anther
culture has many advantages since it does not imply the
inbreeding depression caused by selfing and does not involve
pollinator genome contamination. Many factors are involved
and interact in the optimization of the anther culture protocol
to produce haploid plants; therefore it is necessary to consider:
pre‐treatment of anthers, genotype effects, the stage of
pollen development, culture conditions and induction of
embryos and regeneration of plants. Anther or microspore
cultures have been found to be the most efficient techniques
for obtaining a large number of haploid plants (De Buyser and
Henry.,et al 1980). Haploid production is extremely important
for crop improvement and eradication of pathogens (Brown
and Thorpe et al., 1995) and dramatically improves the yield.
(Badoni and Chauhan et al., 2010). Anther or microspore
cultures has proved to be the most efficient techniques for
obtaining a large number of haploid plants (Buyser and Henry
et al., 1980) and valuable in plant breeding and genetics
studies (Custódioa et al., 2005). The establishment of
homozygous lines of new varieties is possible in a short
period of time, hence provide a useful tool to accelerate plant
breeding successions (Devaux and Li et al., 2001; Zhang et
al., 2011). Haploids can obtained through natural
parthenogenesis or androgenesis. Artificial induction of
ovaries (Muren et al., 1989), ovules (Hansen et al., 1994),
anthers (Foroughi et al., 1982), microspores (Kohler and
Wenzel et al., 1985) is also a promising tool for haploid
production.
In the genus Musa, accurate determination of the
ploidy by phenotypic traits, including stomatal size, density,
and pollen size, and by chromosome counting is difficult and
laborious, mainly due to strong genotypic influences (Hamill,
1992; Vandenhout et al., 1995; Van Duren et al., 1996;
Dolezel, 2004). Musa paradisiaca cv. Puttabale (AB group)
belongs to family Musacae and an indigenous banana cultivar
of Karnataka, India. Previously we published the high
frequency plant regeneration protocol from shoot tip, leaf and
immature male floral explants. No reports available on
haploid production from this plant. This paper deals with the
in vitro haploid plant regeneration from anther culture of
banana cultivar Puttabale.
II. MATERIALS AND METHOD
Explants Collection And Sterlization
Banana cv puttubale inflorescence of appropriate
stage was collected from farmyard of Shimoga district,
Karnataka. The inflorescence were disinfected in 2% sodium
hypochloride solution for 15 min, followed by 8-10 rinses in
sterile distilled water.The immature male flowers are detached
from bract, tepals aseptically. Male flowers contained five
stamens with fully developed anthers. Anthers from the
stamens was carefully isolated and transfered into pertriplates.
Anthers with uninucleated microspore stage was determined
using 1% aceto-carmine stain at this stage anthers were
inoculated on to the callusing media.
Plant Regeneration
B

The callusing media consisted of MS media
constituents agumented with 40 g/l sucrose, 8 g/l agar,
160 mg/l adenine sulfate, 100 mg/l tyrosine and the growth
regulator 2, 4-D tested at the range of 1 to 6 mg/l. The
pH was adjusted to 5.8 before autoclaving at 121ºC for
20 min. The cultures were incubated at 25 ± 2ºC with
12 hrs photoperiod and the callogenic frequency
was evaluated after 30 days of incubation. Regenerated
androgenic embryos like structures developed from callus
were transferred to proliferation medium containing different
conentrations of BAP (2 to 6 mg/l) and IAA( 0.2 to 0.6 mg/l)
the media was agumented with 100mg/l of adanine sulphate
and tyrosin. The well grown elongated shoots were
aspectically transferred to the medium containg 0.25 to
o.75mg/l NAA and without hormones fortified with 0.2%
activated charcoal for the proper development of root system.
The plantlets from culture bottles are moved from the
laboratory later they are shifted to green house for primary
hardening where they are first gently washed free of agar
medium. The well rooted plants were washed with 0.2% of
bovastin solution then followed by primary hardening in
cocopeats, maintained in polythene house for a period of two
weeks. During this period, 90-95 % humidity is maintained
for the initial 6-8 days under diffused light, later they were
maintained at 70 %, humidity and 26 °C Then transferred to
green house for secondary hardening in a polythene bags
containing red soil, sand and cattle manure in the ratio of
1:1:2 respectively. The regenarents grew to the height of 10
to 15 cm were transferred to field condition.
Determination of Ploidy Level
The flow cytometric technique was followed to
determine the ploidy level of the anther-derived plants. Leaf
pieces (1×1 cm) from in vitro plants derived from anther
cultures were chopped with a razor blade in 600 μl buffer
solution consisting of 45 mM MgCl2, 30 mM Tri-sodium
citrate (Na3C6H5O7, 2H2O), 20 mM MOPS, 1% triton X-
100, 10 mM sodium-metabisulfite (Na2S2O5) (pH 7) to
obtain a nuclei suspension. The whole sample was sieved
through a 40-μm mesh nylon filter and stained with 6 μg ml-1
DAPI solution. A diploid parental plant was used as an
internal standard. Nuclei analysis was carried out by using
Partec CA II flow cytometer following the method of Assani
et al., 2003.
III. RESULTS AND DISCUSSION
The anthers isolated aseptically were inoculated on to
the callusing media (1 mg/l. 2,4D to 6 mg/l. 2,4D) showed
callogenic response within a week of incubation. Prior to
inoculation anthers were selected at the diod or tetraoid stage
of meiosis (Fig A) Optimum callogenesis noticed at the
concentration of 3 mg/l. 2,4D (Table.1). On callusing media
anthers began to swell and the size increases to three folds.
After ten days of incubation, callogenic mass noticed from the
dorsal side while the anther wall cells turns black (Fig B).
After 20 days of culture organization of embyronic nodules
were appeared from the sprouted callus mass. The formation
of androgenic embryroids from the protruded callus was
similar to the findings of Assani et al., 2002.
The interaction of BAP at higher levels (2.0 – 6.0
mg/l) and IAA at lower levels (0.2 ‐ 0.6 mg/l) provoked the
growth and development of the enbryonic mass in to shoot
buds. The mean number of shoots organized at each
combination of BAP and IAA is shown in the (Table 1). The
shoot multiplication was optimized at the concentration of 4
mg/l BAP and 0.4 mg/l IAA with a mean of 7.10±0.88 shoots
per callus. shoot buds. The caulogenic mass with multiple
shoots were subjected to colchecin treatment with a range of
(0.025% and 0.05%). Then they were transferred to the
development media consisted of 0.6 mg/l NAA and 0.2%
activated charcoal. The well rooted shoots were maintained
on the media for two to three cycles, then they were
transferred to the rhizogenic media.
The induction of roots depends on the composition of
mineral nutrients and growth regulators. Shoot buds were
transferred to MS with 0.6 mg/l NAA, 0.2% activated
charcoal initiated roots from the base of microshoots ( Fig E),
after three weeks of culture. The well rooted plantlets (Fig H)
were hardened primarily in coco peats containing equal
proportion of garden soil, sand, and cattle dung manure in
polythene house for a period of two weeks (Fig I) and then
they were subjected to secondary hardening in perforated
polythene bags in green house (Fig J). The presence of diploid
plants was also observed among the regenerants. While this
could be a consequence of the regeneration of diploid anther
tissues such as anther wall or connective tissue, in monocots
the regeneration of somatic anther tissue has been very rarely
reported. The other possibility is the spontaneous
chromosome doubling in haploid cells. The possible factors
leading to spontaneous doubled-haploid plants could be
nuclear fusion in the early divisions of the microspores,
endomitosis, endoreduplication or multipolar mitosis during
the callus phase (Chen et al.1982; De Buyser and Henry1986).
The polar transport of auxin is essential for the establishment
of bilateral symmetry during embryogenesis in
dicotyledonous and monocotyledonous species (Fisher,c, et
al.1996).
Androgenesis has been traditionally divided in two
main stages, namely induction and regeneration. In the former
one, somatic cells acquire embryogenic characteristics by
means of a complete reorganization of the cellular state,
including physiology, metabolism and gene expression
(Feher, A. et al. 2002). In the present study also androgenic
embryoids developed from the primary callogenic mass and
upon subculture on the cytokinin supplemented media
embryoids grew up in to plantlets.
Many authors have reported that the developmental
stage of anthers can drastically affect plant regeneration
because microspores respond only at a specific development
stage, which ranges from the tetrad, early and mid-uninucleate
to the early binucleate stage. As such anther of the Lilium ×

‘Enchantment’, excised at the uninucleate microspores stage
could form callus and regenerate bulblets (Niimi et al., 2001).
Medium is another important factor affecting anther culture.
Custódio et al. (2005) reported that the best medium for calli
induction of carob tree was MS media supplemented with 2.3
μM 2, 4-D + 8.2 μM TDZ. Plant regeneration from anther of
banana (Assani et al., 2003) was obtained in 4.4 μM BAP +
2.3 μM.
While genotype or days in vitro are factors that favor
the frequency of appearance of DNA polyploid plants or even
DNA aneuploids (Tremblay et al. 1999; Endemann et al.
2001; Currais et al. 2013), Therefore, the changes observed in
nuclear DNA content might be caused by the effect of growth
regulators used in the culture media. We noticed that synthetic
auxin 2, 4-D might be involved in embryoid formation from
the microspore mass, because it was found to be a factor
favouring changes in the genome of in vitro regenerated plants
(May and Sink 1995; Jin et al. 2008; Clarindo et al. 2008).
Additional assays would be useful to confirm the possible role
of 2,4-D as a factor promoting the variations observed, as well
as NAA or BAP, which were also mentioned as possible
mutagenic agents (Lim and Loh 2003; Mishiba et al. 2006;
Barow and Jovtchev 2007).
The histogram developed from the result of
cytometric analysis (Fig. 2 .A and B) showed a prominent
peak of nuclei in PI fluorescent intensity in Gi l and G1 stages
of interphase of the doubled diploid cells. The intensity of the
nuclei in the interphase of doubled diploid plant and it is two
folds as compared to haploid cells. Extensive fluorescent
emissions at higher intensities are the indicative of
populations of nuclei at increased ploidy levels. Flow
cytometric analyses was performed by measuring the leaf
DNA intensity of the in vitro regenerated plants. The report
of Jin et al. (2008) suggested that detection of plants with
lower nuclear DNA intensity value is due to the existence of
aneuploid plants. This assumption is based on the fact that
many of the changes that can be detected by FCM are
associated with variations in the number of chromosomes
(Clarindo et al. 2008; Currais et al. 2013). These variations
might be enough to indicate the complete or partial loss of a
chromosomes during doubling process (Pfosser et al. 1995;
Roux et al. 2003).The flow cytometric studies enlightened
many investigators to detect plants with values of nearly
twice the nuclear DNA content, suggesting a possible in vitro
polyploidization (Barow and Jovtchev 2007).
IV. CONCLUSION
Anther culture is the classical method employed in
the regeneration of haploid plants but in conventional
breeding production of haloid plant require screening of many
generations and it is difficult in pseudo fruit crops like banana.
The results of the investigation reported here showed the
efficient production haploid plants from the anther culture of
Musa paradisica cv. Puttabale
Fig. 1 High frequency regeneration of plantlets from anther explants of Musa
paradisica cv. Puttabale.
A. Uninuleate stage of anther. B. Development of anther callus. C and D.
Androgenic embryos formed from the calli. E and F. Shoot growth of the
embroids. G. Growth of haploid plants. H. Root induction from the haploid
plants. I. Primary hardening plants. J. Secondary hardened haploid plants.
A B
C D
E F
G H
I J

Figure 2
Histograms of relative DNA content obtained after flow cytometric analysis of nuclei isolated from (A) leaf tissue of the diploid banana plant, and (B) leaf tissue
of the haploid banana plant
Table 1 Effects of growth regulators on frequency of anther callogenesis and plant regeneration from Musa paradisiaca cv.
Puttabale
Plant growth regulators mg/L Frequency of callus formation per
explant
%
Number of multiple shoots per propagule
Mean ± S.D
2, 4 -D mg/L BAP mg/L IAA mg/L
1
2
3
4
5
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
F Value
-
-
-
-
-
-
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
-
-
-
-
-
-
0.2
0.4
0.6
0.2
0.4
0.6
0.2
0.4
0.6
0.2
0.4
0.6
0.2
0.4
0.6
16.66
36.66
90.00
56.66
43.33
33.33
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.90±0.74
1.60±0.70
1.30±0.95
2.00±0.47
3.00±0.67
2.10±0.57
3.20±1.14
7.10±0.88
4.40±1.71
4.00±1.15
5.50±0.97
4.60±1.35
3.30±1.16
5.40±1.58
4.20±1.03
27.3
The value of combination consisted of mean ± S.D. of 10 replicates.
The F-value is significantly different when p< 0.05.

REFERENCES
[1]. Assani , F. Bakry ,F. Kerbellec, R. Haicour G. Wenzel, B.
Foroughi-Wehr (2003) Afza R, Shen M, Zapata J, Xie J, Fundi H,
Lee K, Bobadilla E, Kodym A(2000)., Effect of spikelet
position on rice anther culture efficiency., Plant Sci. 153:155-159.
[2]. Assani A, Bakry F, Kerbellec F, Haicour R, Wenzel G, Foroughi
B (2003)., Production ofhaploids from anther culture of banana
[Musa balbisiana (BB)]., Plant Cell Rep., 21: 511-516.
[3]. Assani A, Haicour R, Wenzel G, Cote FX, Bakry F, Foroughi-
Wehr B, Ducreux G, AguillarME, Grapin A (2001) Plant
regeneration from protoplasts of dessert banana Grande Naine
(Musa spp., Cavendish sub-group AAA) via somatic
embryogenesis. Plant Cell Rep 20:482–488.
[4]. Assani A, Haïcour R, Wenzel G, Foroughi-Wehr B, Bakry F, Côte
FX, Ducreux G, Ambroise A, Grapin A (2002) Influence of donor
material and genotype on protoplast regeneration in banana and
plantain cultivars (Musa spp.). Plant Sci 162:355–362.
[5]. Barow M, Jovtchev G (2007) Endopolyploidy in plants and its
analysis by flow cytometry.In: Dolezel J, Greilhuber J, Suda J
(eds) Flow cytometry with plant cells. Wiley,
Weinheim,Bronsema F, Van O, Prinsen E, Van L(1998).,
Distribution of (14C) dichlorophenoxyacetic acid in cultured
zygotic embryos of Zea mays L., JPlant Growth Regul.,17:81–88.
[6]. Barow M, Jovtchev G (2007) Endopolyploidy in plants and its
analysis by flow cytometry. In: Dolezel J, Greilhuber J, Suda J
(eds) Flow cytometry with plant cells. Wiley, Weinheim, pp
349–372.
[7]. Chen Z, Qian C, Qin M, Xu X, Xiao Y (1982) Recent advances in
anther culture of Hevea brasiliensis (Muell.-Arg.). Theor Appl
Genet 62:103–108.
[8]. Clarindo WR, de Carvalho CR, Arau´jo FS, de Abreu IS, Otoni
WC (2008) Recovering polyploid papaya in vitro regenerants as
screened by flow cytometry. Plant Cell Tissue Organ Cult 92:207–
214.
[9]. Currais L, Loureiro J, Santos C, Canhoto JM (2013) Ploidy
stability in embryogenic cultures and regenerated plantlets of
tamarillo. Plant Cell Tissue Organ Cult 114:149–159.
[10]. Custódioa L, Carneirob MF, Romanoa A (2005).
Microsporogenesis and anther culture in Ceratonia siliqua L.).
Scientia Horticulturae, 104: 65-77.
[11]. De Buyser J, Henry Y (1980) Induction of haploid and diploid
plants through in vitro anther culture of haploid wheat (n=3x=21).
Theor Appl Genet 57:57–58.
[12]. De Buyser, J. and Henry, Y., Wheat production of haploids,
performance of doubled haploids, and yield trials. Biotechnology
in Agriculture and Forestry, Transgenic Crops I (ed. Bajaj, Y. P.
S.), Springer–Verlag, Berlin, 1986, vol. 2, pp. 73–88
[13]. Devaux H, Li P (2001). Enhancement of microspore culture
efficiency of recalcitrant barley genotypes 20: 475.
doi:10.1007/s002990100368
[14]. Dolezel and Batros,.J. Dolezel, J. Batros (2005) Plant DNA flow
cytometry and estimation of nuclear genome sizeAnnals of
Botany, 95 (2005), pp. 99–110.
[15]. Endemann, M., K. Hristoforoglu, T. Stauber and E. Wilhelm.
2001. Assessment of age-related polyploidy in Quercus robur L.
somatic embryos and regenerated plants using DNA flow
cytometry. Biol. Plant. 44:339–345.
[16]. Feher A, Pasternak T, Otvos K, Miskolczi P, Dudits D(2002).,
Induction of embryogenic competence in somatic plantcells: a
review., Biol Bratislava.,57:5–12.
[17]. Fisher C, Neuhaus G (1996)., Influence of auxin on the
establishment of bilateral symmetry in monocots., Plant J.,9:659–
669.
[18]. Foroughi-Wehr B, Friedt W, Wenzel G (1982) On the genetic
improvement génétique du bananier (Musa spp.). PhD thesis,
Ecole Nationale Agronomique de Rennes, France Jin S, Mushke
R, Zhu H, Tu L, Lin Z, Zhang Y, Zhang X (2008) Detection of
somaclonal variation of cotton (Gossypium hirsutum) using
cytogenetics, flow cytometry and molecular markers. Plant Cell
Rep 27(8):1303–1316.
[19]. Hamill, S.D. Hamill, M.K. Smith, W.A.(1992) Dodd In vitro
induction of banana autotetraploids by colchicine treatment of
micropropagated diploids Australian Journal of Botany, 40 (1992),
pp. 887–896.
[20]. Hansen AL, Plever C, Pedersen HC, Keimer B, Andersen SB
(1994) Efficient in vitro chromosome doubling during Beta
vulgaris ovule culture. Plant Breed 112:89–95.
[21]. Jin S, Mushke R, Zhu H, Tu L, Lin Z, Zhang Y, Zhang X (2008)
Detection of somaclonal variation of cotton (Gossypium
hirsutum) using cytogenetics, flow cytometry and molecular
markers. Plant Cell Rep 27(8):1303–1316.
[22]. Kerbellec F (1996) Etablissement d’une technique d’androgenèse
pour l’amélioration génétique du bananier (Musa spp.).
[23]. Kohler F, Wenzel G (1985) Regeneration of isolated barley
microspores in conditioned media and trials to characterise the
responsible factor. J Plant Physiol 121:181–191.
[24]. Lim WL, Loh CS (2003) Endopolyploidy in Vanda Miss
Joaquim (Orchidaceae). New Phytol 159:279–287.
[25]. May RA, Sink KC (1995) Genotype and auxin influence direct
somatic embryogenesis from protoplasts derived from
embryogenic cell suspensions of Asparagus officinalis L. Plant
Sci108:71–84.
[26]. Mishiba K, Tawada K, Mii M (2006) Ploidy distribution in the
explant tissue and the calluses induced during the initial stage of
internode segment culture of Asparagus officinalis L. In Vitro
Cell Dev Biol Plant 42:83–88.
[27]. Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 15:473–
497.
[28]. Muren RC (1989) Haploid plant induction from unpollinated
ovaries in onion. Hortic Sci 24:833–834.
[29]. Niimi Y, Han D, Fujisaki M (2001)., Production of virus-free
plantlets by anther culture of Lilium×‘Enchantment’., Scientia
Horticulturae.,90: 325-334.
[30]. Pfosser M, Amon A, Lelley T, Heberle-Bors E (1995) Evaluation
of sensitivity of flow cytometry in detecting aneuploidy in wheat–
rye addition lines. Cytometry 21:387–393.pp 349–372.
[31]. Roux N, Toloza A, Radecki Z et al (2003) Rapid detection of
aneuploidy using flow cytometry. Plant Cell Rep 21:483–490.
[32]. Tenhola T, Tanhuanpä P, Immonen S (2005)., The effect of cold
and heat treatments on the anther culture response of diverse rye
genotypes., Euphytica.,145: 1-9.
[33]. Tremblay L, Levasseur C, Tremblay F M (1999) Frequency of
somoclonal variations in plant of black spruce (Picea mariana)
and white spruce (P.glauca, pinaceae) derived from somatic
embryogenesis and identification of some factors involved in
genetic instability.Am J Bot86:1373- 1381
[34]. Van Duren et al., M. Van Duren, R. Morpugo, J. Dolezel, R.
Afza.(1996) Induction and verification of autotetraploids in
diploid banana (Musa acuminata) by in vitro techniquesEuphytica,
88 (1996), pp. 25–34.
[35]. Vandenhout et al.,H. Vandenhout, R. Ortiz, D. Vuylsteke, R.
Swennen, K.V. Bai(1995)Effect of ploidy on stomatal and other
quantitative traits in plantain and banana hybrids Euphytica, 83
(1995), pp. 117–122.

ANTHER CULTURE

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to ANTHER CULTURE

Similar to ANTHER CULTURE (20)

More from RSIS International

More from RSIS International (20)

Recently uploaded

Recently uploaded (20)

ANTHER CULTURE